The present invention relates to a process of purification to homogeneity of ATP-diphosphohydrolases involved in numerous nucleotide and nucleoside receptor-mediated physiological functions, namely platelet aggregation, vascular tone, secretory, inflammatory and excretory functions and neurotransmission. These enzymes, which have been particularly obtained from bovine aorta and pig pancreas have been purified and their catalytic unit identified. The partial amino acid sequences of each ATPDase show a high degree of homology with a lymphoid cell activation system named CD39.
ATP-diphosphohydrolases (ATPDases) or apyrases (EC 3.6.1.5) have been found in plants, invertebrates and vertebrates. The enzyme catalyses the sequential hydrolysis of the xcexd- and xcex2-phosphate residues of triphospho- and diphosphonucleosides. These enzymes are generally activated in the presence of divalent cations Ca+2 or Mg+2 and inhibited by sodium azide. In plants, the enzymes are found in the cytoplasm, in soluble or membrane-associated forms, and are generally more active at acid pH. Their precise function is not known, but there is some evidence that they are involved in the synthesis of carbohydrates. In invertebrates, the enzymes are more active at neutral or alkaline pH. Found mainly in saliva and in salivary glands of hematophagous insects, an antihemostatic role has been demonstrated. In vertebrates, a limited number of studies have already defined a diversity of ATPDases. The catalytic site of these enzymes is generally exposed to extracytoplasmic spaces (ectoenzymes). By their location and kinetic properties, these different types of ATPDases could influence the main systems of the organism, namely vascular and nervous systems. Their specific role in these systems is determined by the presence of purine and pyrimidine receptors which react with triphosphonucleosides and their derivatives at the surface of numerous cell types.
Presence of both ectoATPase and ectoADPase activities in the vascular system has been known for many years, and up until the work of Yagi et al. (1989), they were attributed to two distinct enzymes. The latter purified these activities and showed that in bovine aorta, a single enzyme was responsible for the sequential hydrolysis of ATP and ADP. A mammalian ATPDase had been first described in the pancreas (Lebel et al., 1980) and was further reported in several other tissues. Yagi et al. (1989) proposed that the enzyme from aorta was similar to the previously reported mammalian ATPDase from pancreas and that it was associated with the intima of bovine aorta. Purification to homogeneity was demonstrated by SDS-polyacrylamide gel electrophoresis (PAGE) and silver staining. The apparent molecular weight of the pure enzyme was estimated at 110 KDa. The existence of the ATPDase in the bovine aorta was corroborated by Cxc3x4txc3xa9 et al. (1991) who, by showing that identical heat and irradiation-inactivation curves with ATP and ADP as substrates, assigned to the same catalytic site the ATPase and ADPase activities. A comparison of the biochemical properties led Cxc3x4txc3xa9 et al. supra to propose that the bovine aorta enzyme was different from the pancreas ATPDase. Indeed, the enzymes have different native molecular weights, optimum pH and sensitivities to inhibitors. They proposed to identify pancreas enzyme as type I and the aorta enzyme as type II. In the bovine aorta, the enzyme was found to be associated with smooth muscle cells and endothelial cells and could inhibit ADP-induced platelet aggregation. Cxc3x4txc3xa9 et al. (1991) further showed that concurrent addition of ATPDase and ATP to platelet-rich plasma resulted in an immediate dose-dependent platelet aggregation caused by the accumulation of ADP, followed by a slow desaggregation attributable to its hydrolysis to AMP. In the absence of ATPDase, ATP did not induce any aggregation while ADP initiate an irreversible aggregation which extent is limited by the ADPase activity of the enzyme. ATPDase also attenuated the aggregation elicited by thrombin and collagen but not by PAF (Platelet Activating Factor), the first two agonists having an effect mediated by platelet ADP release. It was therefore suggested that ATPDase had a dual role in regulating platelet activation. By converting ATP released from damaged vessel cells into ADP, the enzyme induced platelet aggregation at the sites of vascular injury. By converting ADP released from aggregated platelets and/or from hemolyzed red blood cells to AMP, the ATPDase could inhibit or reverse platelet activation, and consequently limit the growth of platelet thrombus at the site of injury. In their attempt to further characterize the aorta ATPDase, the present inventors have developed a new process for producing highly purified ATPDases. They have established a procedure by which its specific activity can be increased over and above the activity of a crude cell preparation by more than 10000-fold. They also discover that the purified enzyme (the catalytic unit) had a molecular weight different from the one previously reported for the native form of the enzyme (190 KD by using the irradiation technique), suggesting that the enzyme may exist in a multimeric form in its native state. Partial amino acid sequences of both bovine aorta and porcine pancreatic ATPases have been obtained.
In a completely different field, Maliszenski et al. (1994) have published the sequence of a human lymphoid cell activation antigen designated CD39. Another group (Christoforidis et al. 1995) described the purification of a human placenta ATPDase of a molecular weight of 82 KDa. Its partial amino acid sequence shows a high degree of homology with CD39.
When the above mentioned partial amino acid sequences were entered in GenBank for verifying the presence of any homologous sequence, complete homology was surprisingly found for some of these fragments with the CD39 gene product. The complete sequences of the ATPDases remain to be obtained. Assuming that CD39 is an up to date unknown ATPDase, a process for producing ATPDases by recombinant technology is now possible, and CD39 can now be used to reduce platelet aggregation and thrombogenicity.
It is an object of the present invention to provide two ATPDases isolated from bovine aorta and porcine pancreas, which enzymes have a molecular weight for their catalytic unit of about 78 and 54 Kilodaltons, respectively. A novel process for obtaining a highly purified ATPDase is also an object of the present invention. This process has been successfully applied to the purification of both the pancreatic and the aorta enzymes and is deemed to work in the purification of any ATPDase. For both sources of enzymes, the process allows the specific activity of the enzyme to be increased by at least 300 fold when compared to the activity retrieved in the microsomial fraction of these cells as previously reported for an aortic and pancreatic proteins of a native molecular weight of about 190 and 130 KDa, respectively.
The two ATPDases purified to homogeneity were partially sequenced. These sequences have shown striking similarities with a human lymphoid cell activation antigen named CD39 (Maliszenski et al., 1994). Since the molecular weight of CD39 and its glycosylation rate appears to define a human counterpart for the present bovine aortic ATPDase, it is the first time that a sequence is assigned to an ATPDase. A process of producing an ATPDase by recombinant technology is now possible using a host cell expressing the CD39 human protein, its homologous sequences in bovine and porcine species, and variants and parts thereof.
The present invention also relates to the use of CD39 and of the above bovine and porcine homologous proteins for reducing platelet aggregation and thrombogenicity.
The research team to which the present inventors belong has already characterized the pig pancreatic ATPDase, and the latter reassessed the properties of the bovine aorta enzyme. They confirmed that the aorta ATPDase was different from its pancreatic counterpart. They have found previously (Cxc3x4txc3xa9 et al., 1992) that the aorta enzyme (isolated from a microsomal fraction of the cells) had a molecular weight of about 190 kDa in its native state. In their work for extensively purify this enzyme, they found that the highly purified enzyme had a molecular weight on SDS-PAGE of about 78 KDa. Yagi et al. (1989) have already shown that an ATPDase purified to homogeneity had a molecular weight of 110 KDa. After purifying the enzyme by the present method, the 110 kDa band was indeed absent from SDS-PAGE. A unique band migrating of an estimated weight of 78 KDa was rather revealed. The confirmation of the identity of the purified enzyme was achieved by binding FSBA, an ATP analog binding the enzyme, to the separated and blotted enzyme. The use of anti-FSBA antibodies revealed the presence of the bound enzyme and this binding was inhibited with ATP and ADP. The same procedure was applied to confirm the identification of the pancreas ATPDase Type I.
The present process allows the purification of ATPDases to a very high level. In the aorta, the purified enzyme has a specific activity which is increased by at least 300 fold compared with the specific activity of microsomal fraction (already enriched by about 30 fold from the crude cell preparation).
The bovine aorta and porcine pancreatic ATPDases have been partially sequenced, and the sequences have been found to be highly homologous to a human lymphoid cell activation antigen designated CD39 (Maliszenski et al.,op. cit.). The complete sequences of the ATPDases types I and II have not been obtained yet. If one assumes that CD39 gene product is an ATPDase type II, the present invention therefore contemplates the use of CD39 in the reduction of platelet aggregation and of thrombogenicity, as well as a process of making ATPDases using the CD39 sequence, variants or parts thereof (recombinant technology).
The present invention will be described hereinbelow with reference to the following Examples and Figures which purpose is to illustrate rather than to limit the scope of the present invention.